Conformational stability of digestion-resistant peptides of peanut conglutins reveals the molecular basis of their allergenicity

Abstract

Conglutins represent the major peanut allergens and are renowned for their resistance to gastro-intestinal digestion. Our aim was to characterize the digestion-resistant peptides (DRPs) of conglutins by biochemical and biophysical methods followed by a molecular dynamics simulation in order to better understand the molecular basis of food protein allergenicity. We have mapped proteolysis sites at the N- and C-termini and at a limited internal segment, while other potential proteolysis sites remained unaffected. Molecular dynamics simulation showed that proteolysis only occurred in the vibrant regions of the proteins. DRPs appeared to be conformationally stable as intact conglutins. Also, the overall secondary structure and IgE-binding potency of DRPs was comparable to that of intact conglutins. The stability of conglutins toward gastro-intestinal digestion, combined with the conformational stability of the resulting DRPs provide conditions for optimal exposure to the intestinal immune system, providing an explanation for the extraordinary allergenicity of peanut conglutins.

Modification of the reaction system of Ara h 2 catalyzed by MTGase: Products and reaction conditions analysis

“…As shown in Figure b, a peak was observed at the molecular weight of approximately 10 kDa (peak b ) after the enzymolysis of Ara h 2 with trypsin. The results were completely consistent with those of Sen et al () who described a 10 kDa peptide after digestion of Ara h 2 with trypsin; moreover, the cleavage site is attributed to Arg 62 –Cys 63 or Arg 92 –Gln 93 (Apostolovic et al, ; Koppelman et al, ). In the black profile, the molecular weight of peak a , which represented an undigested Ara h 2, was about 17 kDa.…”

“…In the black profile, the molecular weight of peak a, which repre- may be hidden, and/or other cleavage sites were exposed. As shown in the spatial structure of Ara h 2.02 (Figure 1b), Arg 62 and Arg 92 were both located in the conformationally vibrant regions (Apostolovic et al, 2016) and nearby Gln 69 and Gln 76 , which resulted in the disappearance of peak 2. Thus, Arg 62 and Arg 92 may be hidden if Lys 52 cross-linked with Gln 69 or Gln 76 .…”

Influence of peanut matrix on stability of allergens in gastric‐simulated digesta: 2S albumins are main contributors to the IgE reactivity of short digestion‐resistant peptides

“…Dried TCA/acetone protein pellets from liquid portion of gastric‐simulated digesta were re‐suspended in Laemmli sample buffer (reducing and non‐reducing conditions). Isoelectrofocusing and 2D SDS‐PAGE were performed as per the method of Apostolovic et al . For more details, see .…”

“…In-gel digestion of 2D SDS-PAGE proteins and nano-liquid chromatography coupled to tandem mass spectrometry (nLC-MS/MS) Protein spots from 2D gels were manually excised and in-gel digested, according to the method of Shevchenko et al14 Obtainedpeptides were analysed, as previously reported method,13,15 with an LTQ Orbitrap XL mass spectrometer and EASY-nLC II system (Thermo Fisher Scientific Inc., Bremen, Germany).…”

Meta‐Analysis for Correlating Structure of Bioactive Peptides in Foods of Animal Origin with Regard to Effect and Stability

“…P was present in 67% of the sequences, and 6.7% of these had PP doublets, which confer stability against serum proteases (Foltz et al., ; Jambunathan & Galande, ). This high percentage of P residues in stable peptides might be the consequence of conformational changes, which reduce affinity of proteases to such peptides (Apostolovic et al., ). However, the position of P depended on peptide length (Figure ).…”